Biological Evaluation of Human Degenerated Nucleus Pulposus Cells in Functionalized Self-Assembling Peptide Nanofiber Hydrogel Scaffold

Abstract

Nucleus pulposus (NP) tissue engineering has been proposed as a novel biological treatment for early-stage intervertebral disc degeneration. In this study, a novel functional self-assembling peptide PKP was first designed by linking the short functional motif of bone morphogenetic protein-7 (BMP7) to the C-terminal of RADA16-I, and another new functional self-assembling peptide was obtained by mixing RKP with RADA16-I. Then, the biocompatibilities and bioactivities of RKP and RAD-RKP for human degenerated nucleus pulposus cells (hNPCs) were studied in vitro. Atomic force microscopy and scanning electron microscopy (SEM) confirmed that both RKP and RAD-RKP could self-assemble into three-dimensional (3D) nanofiber hydrogel scaffolds in a culture medium at 37°C. After the hNPCs were cultured in 3D scaffolds, both RKP and RAD-RKP exhibited reliable attachment and extremely low cytotoxicities (<14%), which were verified by SEM and cytotoxity assays, respectively. Our results also showed that the functional-based scaffolds could increase the proliferation and migration of hNPCs after 7 days compared with culture plates and pure RADA16-I. Quantitative real-time polymerase chain reaction demonstrated that the expressions of collagen II α1, Sox-9, and aggrecan were upregulated, while collagen I α1 was downregulated by functional-based scaffolds after 28 days. Furthermore, we also confirmed that RAD-RKP exhibited a higher hNPC proliferation, migration, and expression of Sox-9 and aggrecan compared with pure RKP. Therefore, the results of this study indicated that the BMP7 short motif-designed functional self-assembling peptide nanofiber hydrogels could be used as excellent scaffolds in NP tissue engineering, and RAD-RKP might have further potential application in human mild degenerated NP tissue regeneration.

Functional self‐assembled peptide scaffold inhibits tumor necrosis factor‐alpha‐induced inflammation and apoptosis in nucleus pulposus cells by suppressing nuclear factor‐κB signaling

“…Total RNA was extracted from cells/scaffold, using TRIzol (Invitrogen) according to the manufacturer's instructions . The RNA concentration was determined by spectrophotometry and RNA was reverse‐transcribed with PrimeScript™ RT Master Mix (TaKaRa, China).…”

“…Our previous studies reported a novel type of functional self‐assembling peptide (RADA‐KPSS) constructed by conjugating short functional motifs of BMP7 (KPSS) to the C‐terminus of the RADA16‐I. It displays three benefits: good biocompatibility, excellent bioactivity, and injectability . Furthermore, we demonstrated that the RADA‐KPSS peptide enhanced NPC proliferation and ECM synthesis in physiological conditions .…”

“…Self‐assembling peptide scaffolds have attracted considerable research attention because of their ability to self‐assemble and form nanofibrous hydrogel scaffolds under physiological conditions, with similar structures as ECM from nucleus pulposus (NP) tissue . Previously, we studied this promising ability of functional self‐assembling RADA‐KPSS peptide scaffolds, using RADA16‐KPSS, which was formed by conjugating BMP‐7 short functional peptide to the C‐terminus of RADA16‐I (a very important member of the self‐assembling peptide family), in tissue regeneration of IDD . However, whether this novel functional self‐assembling peptide scaffold has protective abilities in NPCs in an inflammatory environment has not been fully investigated.…”

BMP7-Based Functionalized Self-Assembling Peptides for Nucleus Pulposus Tissue Engineering

“…35 Only when KPS was mixed with RADA16-I at an equal volume ratio (RAD-KPS) were the β-sheet structures regained. Furthermore, our previous study also showed that RAD-KPS was more suitable for NP tissue engineering applications than was pure RKP.…”

“…31,32 BMP7 not only stimulated nucleus pulposus cells to secrete aggrecan and collagen II in vitro but also prevented disc degeneration in vivo. 31 35 we successful conjugated one important short peptide, KPS, to the C-terminus of RADA16-I to obtain a novel functionalized self-assembling peptide RKP and found that it could be used to form excellent scaffolds for NP tissue engineering when mixed with RADA16-I at an equal volume ratio. However, BMP7 contains three different important short functional motifs, as above-mentioned.…”